Fermentation process for the production of 2-ketogluconic acid



Patented Mar. 31 1942 'FERMENTATION rnoonss Fon'r m PRO- DUCTION orz-xn'roowcomc ACID Lewis B. Lockwood, Alexandria, and George E. Ward,Arlington, Va., and Joseph J. Stubbs, Washington, D. C., and Edward T.Roe, Arlington, and Benjamin Tabenkin, Alexandria, Va., assignors toHenry A. Wallace, as Secretary of Agriculture 01' the United States ofAmerica, and to his successors in ofli'ce No Drawing. Applicaiign July8, 1940, Serial No. a 3 o i 7 Claims. (01. 195-47) (Granted under theact of March 3, 1883, as amended April 30, 1928; 370 0. 757) Thisapplication is' made under the act of March 3, 1883, as amended by theact of April 30, 1928, and the invention herein described and claimed,if patented, may be manufactured-and used by or for the Government ofthe United States of America for governmental purposes without thepayment to us of any royalty thereon;

This invention relates to a method for the preparation of ketocarboxylicacids, and more particularly to a method for the preparation, by

fermentation, of a valuab1e.material which finds use as an intermediatefor the preparation of d- -isoascorbic acid (also known asd-araboascorbic acid), which possesses marked antiscorbutic andantioxidant properties. (Maurer and Schiedt, Ber. der deutsch. chem.Gesell. vol. 66, page 1054 (1933.) and vol. 67, page 1239 (1934); alsoOhle, U. S. Patent 2,160,621 (1939); also Dalmer and Moll, Zeitschr.'physiol, Chemie, vol. 222, page The production of 2*ketog1uconi'c acidby fermentation methods has been only recently observed. Thus, in 1935,Bernhauer and Giirlich (Biochemlsche Zeitschrift 280, 357) isolatedsmall quantities of 2-ketogluconic acid'from cultures of Acetobactergluconicum grown on solutions of calcium gluconate, and in 1938,Bernhauer and -Knobloch (Naturwissenschaften 50,819) obtained2-ketogluconic acid from cultures of Acetobacter suboxydans grown onaqueous solutions of salts fermentation periods of 20 days or more, itis possible, by applying our invention, to obtain 80 to 95% yields of2-ketogluconic acid in 30 to 40 hours. vA further advantage of ourinvention is that we are not restricted to the use of gluconate salts assubstrates, as heretofore, but we may use glucose or carbohydratematerials containing glucose as a. component of the sugar molecule, suchas maltose, dextrins, starch, molasses, grain mashes, and the like. Astill further advantage of our invention is to be found in the fact thatno' -ketogluconic acid is formed, as has been heretofore encountered.

We have found that numerous bacteria of the genus Pseudornonas possessthe ability to produce 2-ketogluconic acid from. the above-mentionedsubstrates, when cultivated according tothe methods herein set forth.Thus, we have found "that many strains distributed among the followofgluconic acid. In both of these cases the bacteria were grown in thinfilms or pellicles in unagitated surface cultures and the conversion ofsubstrate to product required twenty days, or more. The reaction wascomplicated by the simultaneous formation of 5-ket0g1uconic acid.

The formation of 2-ketogluconic acid was-also erably at superatmosphericpressure, thesystem at the same. time being agitated by various means, arapid and eiiicient. conversion of substrate to Z-ketogluconic acidoccurs. In contrast to prel at super-atmospheric pressure.

ing species may be used to effect this conversion: Pseudomonasschuylkilliensis, Ps. putida, Ps. graveolens, Ps. vendz'elli, Ps.ovalis, Pajragii, Ps. mildenbergii, Ps. mucidolens, and Ps. fluorescens,It is thus apparent that the ability to produce 2-ketogluconic acid ischaracteristic of the genus Pseudomonas taken as a whole, when thebacteria are cultivated in the manner taught by us.-

' In our invention, agitation of the system may be effected by blowingair through the mass, or by propellers, or by revolving the fermenter,or by other means which will occur to those skilled in the art. Theexact apparatus used to effect agitation is not critical, the importantfactor being the intimate contacting of the bacterial cells, ,thesubstrate, the neutralizing agent, and the gas used for aeration.

We have found that aeration of the mash is necessary to obtain a rapidoxidation of the substrate to 2-ket'ogluconic acid. Such aeration may beapplied at atmospheric pressure, or preferably, We have found thatoperating at super-atmospheric pressure re-' sults in a more rapidconversion of substrate to product. I

We have found it desirable to conduct our fermentation at temperaturesbetween 20 C. and C., the range from 25 C. to 30 C. being especiallysuitable.

vious mediocre yields of 2-ketogluconic 3 acid in 50 As typicalapparatus within which our process vention as to equipment used, sincenumerous modifications and adaptations are possible, and will be readilyapparent to those skilled in the art.

Considerable latitude is possible in the selection of nutrients to beused in performing our invention. Although we prefer touse corn steepingliquor, urea, magnesium sulfate, and potassium phosphate, as citedhereatfer in Example 1, it is possible to secure good results if some ofthese components are omitted or varied as to the quantity used. Thus, anentirely satisfac- .tory fermentation may be obtained upon omitting theurea, magnesium sulfate and potassium phosphate, if the quantity of cornsteeping liquor be increased. Similarly, if urea and the abovementionedinorganic salts be supplied in the quantities given in Example 1, thecorn steeping liquor may be reduced in quanity or even entirelyeliminated without noticeably affecting the fermentation. We accordinglydo not wish to be restricted as to the nutrient components, since manyvariations will be apparent to those skilled in the art. Likewise, we donot wish to'be restricted as to the use of calcium carbonate as theneutralizing agent, since the substitution of quicklime, zinc carbonate,and other similar substances will readily occur to those skilled in theart. 1

The following examples illustrate representative procedures used inpracticing our invention:'

Example 1 An aqueous fermentation medium of the following compositionwasused:

' Grams per liter of medium Glucose 100. Corn steeping liquor- 5.Octadecyl alcohol 0.3 Urea 2.0 MgSO4.7H2O -l 0.25 KHzPOi 0.60 CaCO: 27.0

3200 cc. of this sterile medium were inoculated with approximately 300cc. of an active culture of Pseudomonas flum'escens and placed in a 1'0-tary drum fermenter. Air at atmosphericfiiressure was passed throughthrough the fermenter at a rate of 1600 cc. per minute, the solution wasagitated by revolving the drum l3 revolutions per minute, and thetemperature of the system was maintained at approximately 25 C. Thecourse of the fermentation was followed by periodic analysis. After 43hours, the glucose was all consumed and 2-ketogluconic acid (calciumsalt) was present in a quantity equivalent to a 72% yield, based on theglucose available. The prod-' not was identified by its opticalproperties and by preparation of the methyl ester (M. P. 174 C.).-

Elramplc 2 The same materials and conditions were used as in Example 1,except that the process was conducted under increased air pressure, agage pressure of 30 pounds per square 'inch being maintained. After 25hours, the glucose was all consumed and 2-ketogluconic acid (calciumsalt) was present in a quantity equivalent to an 81% yield based on theglucose available.

Example 3 The same materials and conditions were used as in Example 2,except that calcium gluconate (50 grains per liter) was used as thesubstrate instead of glucose. After hours, a 63% yield of 2-ketogluconicacid (calcium salt) was obtained.

Example 4 The same materials and conditions were used as in Example 2,except that potassium gluconate (50 grams per liter) was used as thesubstrate. After 65 hours a 61.5% yield of 2-ketogluconic acid wasobtained.

Example 5 A fermentation medium of the composition given in Example 1was distributed in 200 cc. portions of Jena glass gas-washing bottles(type 1010). Following sterilization, individual bottles were inoculatedwith selected species of Pseudomonas. Each bottle was then aerated for 8days with sterile, humidified air at a rate of approximately 200 cc. perminute, the incubation temperature being 30 C. throughout. The speciesused, and the yields of 2-ketogluconic acid (calcium salt) obtained wereas follows:

-, ,32 liters of a fermentation medium of the composition given inExample 1 was sterilized inavertical aluminum vat equipped withagitating means, and means for dispersing air. The octadecyl alcoho],supplemented by small portions of lard oil, eliminated frothing andfoaming during the fermentation. The nutrient solution was inocu-' latedwith an active culture of Pseudomonas fluorescens and maintained at 25C., with agitation as'above described, and aeration at 12 to 16 iitersof air per minute. The system was maintained at 30 pounds gage pressure.After 43 hours the glucose was'all consumed and an 88% Having thusdescribed our invention, what we claim for Letters Patent is:

1. A process for the production of 2-ketoglu conic acid, which comprisesinoculating a carbohydrate mash with bacteria of the genus Pseudomonas;thence aerating and agitating the inoculated mash, the while cultivatingthe bacteria in a submerged state, thereby producing Z-ketogluconicaci'd.

2. A process for the production of 2-ketoglu Yield conic acid, whichcomprises inoculating a carbohydrate mash with bacteria of the genusPseudomonas, thence aerating and agitating the inoculated mash, .thewhile maintaining the same under super-atmospheric pressure of gasescontaining substantial quantities. of oxygen, and cul Pseudomonas,thence aerating and agitating the inoculated mash, the while maintainingthe same under superatmospheric pressure of gases containing substantialquantities of oxygen, and cultivating the bacteria in a submerged state,thereby producing 2-ketogluconic acid.

5. A process for the production of 2-ketogluconic acid, which comprisesinoculating a nutri-* ent solution containing a gluconate salt withbacteria of the genus Pseudomonas; thence aerating and agitating theinoculated mash, the while cultivating the bacteria in a submergedstate,

thereby producing Z-ketogluconic acid.

6. A process for the production of 2-ketogluconic acid, which comprisesinoculating a nutrient solution containing a gluconate salt withbacteria of the genus Pseudomonas, thence aerating and agitating theinocu1ated mash, the while maintaining the same under super-atmosphericpressure of-gases containing. substantial quantitles of oxygen, andcultivating the bacteria in a "submerged state, thereby producing2-ketogluconic acid.

f'l. A process for the production of 2-ketogluconic acid, whichcomprises inoculating a carbohydrate mash with bacteria of the groupconsisting of the species Pseudomonas fluorescens, Ps. fragii, Ps.graveolens, Ps. mildenbergii, Ps.

' mucidolens, Ps. putida, Ps. schuylkilliensis, Ps.

vendrelli, and Ps. ovalis; thence aerating and agitating the inoculatedmash, the while cultivating the bacteria in a submerged state, therebyproducing2-ketogluconic acid,

' LEWIS B. LOCKWOOD.

GEORGE E. WARD. JOSEPH J. STUBBS. EDWARD T. ROE; BENJAIVHN TABENKIN.

